West Nile (“WN”) virus was first isolated over 60 years ago from the blood of a febrile patient (Smithburn et al., 1940), and is one of the most widespread flaviviruses worldwide. The WN virus is endemic to Africa and has been repeatedly known in Europe and Asia for decades causing self-limiting epidemics and epizootics (Murgue et al., 2001; Savage et al., 1999). Recent introduction of the WN virus into the North American continent (Lanciotti et al., 1999) had disastrous consequences both for wildlife and human population (Roehrig et al., 2002), and in a few years, WN has developed into a nationwide epidemiological problem (CDC, 2005). In humans, WN infection is often inapparent or occurs as a mild febrile disease (Monath and Heinz, 1996). However, the WN virus has also associated with severe neurological symptoms (Flatau et al., 1981; Smithburn et al., 1940), and recent outbreaks of WN infection have been characterized by an increased CNS involvement (Roehrig et al., 2002; Solomon and Vaughn, 2002).
Based on serological data and genetic characterization, WN viruses have been grouped into at least two distinct lineages (Berthet et al., 1997; Price and O'Leary, 1967). Representatives with moderate and high virulence have been found in both WN virus groups (Beasley et al., 2002). Although highly related to certain strains circulating in the Middle East, the NY99 strain is perhaps the most pathogenic and virulent WN strain known to date (Monath, 2001). Recent studies have shown that mice that succumb to encephalitis after peripheral inoculation of NY99 in very small doses (Beasley et al., 2002).
The virions of the WN fever virus are spherical particles with a diameter of 50 nm constituted by a lipoproteic envelope surrounding an icosahedral nucleocapsid containing a positive polarity (+), single-strand RNA. A single open reading frame (“ORF”) encodes all the viral proteins in the form of a polyprotein. The cleaving and maturation of this polyprotein leads to the production of about ten different viral proteins. The structural proteins are encoded by the 5′ part of the genome and correspond to the nucleocapsid designated C (14 kDa), the envelope protein designated E (50 kDa), the pre-membrane protein designated prM (23 kDa), and the membrane protein designated M (7 kDa). The non-structural proteins are encoded by the 3′ part of the genome and correspond to the proteins NS1 (40 kDa), NS2A (19 kDa), NS2B (14 kDa), NS3 (74 kDa), NS4A (15 kDa), NS4B (29 kDa), and NS5 (97 kDa).
In the mouse model, in which flaviviruses are inherently neurovirulent, both neurovirulence and neuroinvasiveness have been positively associated with determinants in the envelope proteins (Cecilia and Gould, 1991; Chambers et al., 1999; Gualano et al., 1998; Hasegawa et al., 1992; Holzmann et al., 1990; Holzmann et al., 1997; Jiang et al., 1993; McMinn, 1997; Pletnev et al., 1992; Pletnev et al., 1993). The envelope protein (E) of many flaviviruses is glycosylated, and while the WN virus is not an exception to this rule, a few non-glycosylated strains have been identified (Beasley et al., 2001; Berthet et al., 1997; Wengler et al., 1985). The importance of E protein glycosylation for expression of the virulent phenotype of lineage I WN viruses has been demonstrated experimentally (Beasley et al., 2005; Shirato et al., 2004). However, evidence documenting negative effects of E glycosylation on the WN virulence in mice or on its infectivity in cell cultures has been reported as well (Chambers et al., 1998; Hanna et al., 2005).
Currently, a number of subunit or recombinant WN vaccines for veterinary and human use are under development (Kahler, 2003; Lai and Monath, 2003; Ng et al., 2003; Nusbaum et al., 2003; Pletnev et al., 2002; Tesh et al., 2002). In contrast to subunit or inactivated vaccines, a live WN vaccine may be expected to elicit a long lasting balanced humoral and cell mediated immune response (Yamshchikov et al., 2005). However, the high virulence and pathogenicity of the NY99 strain (Beasley et al., 2002; Roehrig et al., 2002) makes it questionable for use in development of a live attenuated WN vaccine. Further, the known association of lineage I strains (such as the NY99 strain) with human and equine outbreaks (Lanciotti et al., 2002) raises a concern about their suitability for vaccine development in general. As such, there remains a need for the development of new live WN virus vaccines.
Recently, several (+) RNA virus studies have relied on the infectious clone methodology, which allows for targeted manipulation of viral genomes. In the “classical approach,” (+) RNA viruses are recovered from cells transfected with synthetic RNA made by in vitro transcription of infectious clone cDNA templates. In a layered DNA/RNA approach, also known as “infectious DNA,” the infectious (+) RNA viruses are recovered directly after transfection of plasmids carrying viral genome cDNA into susceptible cells. Unfortunately, difficulties are often encountered in the design of flavivirus infectious DNA. In addition, few studies have reported on the use of such infectious DNA constructs as a vaccine. Recently, applicant developed an infectious DNA construct encoding an attenuated WN virus denominated as WN1415 as described in co-pending patent application Ser. No. 11/065,783, which is incorporated by reference. Although the infectious DNA construct and virus may be useful as an immunogenic composition for vaccination against WN, more efficacious immunization regimens are desired by increasing the antigenic similarity of vaccines to the circulating NY99 strain.